Nematode biopesticide

ABSTRACT

A composition and method for controlling scarabs, especially lawn scarabs, utilising certain strains of the entomopathogenic nematode species,  Heterorhabditis zealandica  are disclosed. The nematode strains used in the composition and method generally have an LD 50  value of less than 300 IJ as measured by pot assays against final instar scarab larvae.

FIELD OF THE INVENTION

The present invention relates to a composition and method forcontrolling scarabs, especially lawn scarabs, utilising certain strainsof the entomopathogenic nematode species Heterorhabditis zealandica.

BACKGROUND OF THE INVENTION

The family of beetles known as Scarabaeidae includes a number of speciesthat are known as significant agricultural and horticultural pests.Larvae of lawn scarabs (such as Cyclocephala signaticollis, Heteronychusarator, Adoryphorus couloni, Antitrogus morbillosus, Anoplognathusporosus, Ataenius imparalis, Sericesthis geminata, S. pruinosus, S.nigrolineata, Scityla sericans, Saulostomus villosus, Aphodiustasmaniae, Heteronyx spp, Rhopoea magnicornis, Popillia japonica,Cyclocephala borealis, C. hirta, C. parallela, Melolontha melolontha,Anomala aenea, Phyllophaga phyllophaga, P. hirticula, Plylloperthahorticola, Haplididia etrusca, Maladea matrida, Costelytra zealandica,Amphimallon solstatialis, and Ligyrus subtropicus) feed on the roots ofgrasses thereby causing considerable damage to pastures, lawns andanmenity turf. Control treatments typically involve the use of chemicalpesticide sprays, however these have a number of disadvantages.including low efficacy (particularly against final instar larvae), lowspecificity and public concern regarding pesticide residues.Consequently, there is a need for a viable alternative to the control oflawn scarabs by chemical pesticide spraying. In this regard, the presentinventors have identified certain strains of the entomopatliogenicnematode species H. zealandica that are suitable for use as biologicalcontrol agents for lawn scarabs.

DISCLOSURE OF THE INVENTION

Thus, in a first aspect, the present invention provides a compositionfor controlling a population of larval and/or pupal scarabs, comprisingan amount of an entomopathogenic nematode optionally in admixture with asuitable agricultural and/or horticultural carrier, wherein saidentomopathogenic nematode belongs to the species Heterorhabditiszealandica and has an LD50 value of less than 300 infective juveniles(IJ) as measured by pot assays against final instar scarab larvae.

Preferably, the entomopathogeniic nematode belongs to a strain of H.zealandica which has an LD50 value of less than 300 IJ as measured bypot assays against final instar Cyclocephlala signaticollis larvaeand/or final instar Popillia japonica larvae.

More preferably, the entomopathogenic nematode belongs to a strain of H.zealandica which has an LD50 value of less than 175 IJ against finalinstar C. signaticollis larvae and/or final instar P. japonica larvae.Especially preferred are the strains designated JB1/X1, GKB and JB3D.

Compositions will include an amount of the entomopathogenic nematodewhich is, typically, about 50 to 10,000, more preferably about 500 to1000, IJ/ml of composition.

Compositions will also typically include a suitable agricultural and/orhorticultural carrier. Where the composition is desired to be in theform of an aqueous spray, the carrier may be selected from, for example,water or solutions in water of polyethylene glycol or glycerol or smallquantities of wetting agent or various substances to stimulate nematodeactivity such as citric acid, insect blood or low concentrations ofchemical pesticide. Where the composition is desired to be in a solidform, the carrier may be selected from, for example, calcium alginateand polyacrylamide (as would be suitable for gelled pellets),attapulgite or vermiculite (as would be suitable for solid granules), orother moist substrates such as peat, sponge, sawdust or cellulose.Compositions in solid form may be dispersed into an aqueous carrier(such as those mentioned above) for use as an aqueous spray.

Compositions are preferably stored at low temperature (e.g. 2 to 10° C.)under aerobic conditions, and at a water activity of abpout A_(w) 0.97.

In a second aspect, the present invention provides a method forcontrolling a population of larval and/or pupal scarabs in an affectedarea, said method comprising applying to said area a composition inaccordance with the first aspect.

For compositions in the form of aqueous sprays, application may becarried out with typical agricultural and/or horticultural sprayingequipment including pressurised, fan sprayers venturi sprays and boomsprayers. For compositions to be applied in a solid form, applicationmay be carried out with typical agricultural and/or horticulturalscattering equipment such as those used for spreading fertilisers onlawn.

The composition will typically be applied to an affected area which hasbeen subjected to heavy watering in amounts sufficient to provide 50,000to 1 million IJ/m², nmore preferably 100,000 to 500,000 IJ/m². Followingapplication, it is also preferable to submit the affected area onceagain to heavy watering in order to soak the composition into the rootzone where the larval scarabs feed. Application of the composition ispreferably conducted at dusk.

The composition and method of the invention may be used for the controlof lawn scarabs (such as those mentioned above) and other pest scarabs(e.g. sugar cane scarabs, blueberry scarabs, etc.)

In a further aspect, the present invention provides a nematode, in asubstantially purified form, selected from the H. zealandica strainsdesignated JB1/X1, GKB and JB3D.

The term “controlling” as used herein in relation to a population oflarval and/or pupal scarabs, is intended to refer to both maintaining(i.e. preventing increases) and reducing said population.

The terms “comprise”, “comprises” and “comprising” as used throughoutthe specification are intended to refer to the inclusion of a statedstep, component or feature or group of steps, components or featureswith or without the inclusion of a further step, component or feature orgroup of steps, components or features.

The invention will hereinafter be described with reference to thefollowing non-limiting examples and accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1 shows a RAPD gel conducted on DNA from H. zealandica and othercomparative strains. The primer used was OP-A04: 5′-AATCGGGCTG-3′ (SEQID NO:1) (Operon Technologies Inc.). (Key: 1. 100 bp DNA Ladder; 2. H.zealandica (Great Keppel A); 3. H. zealandica (Great Keppel B); 4. H.zealandica (Great Keppel C); 5. H. zealandica (Windsor); 6. H.zealandica (NZH3); 7. H. zealandica (WA Het); 8. Heterorhabditis sp.(JB6); 9. H. zealandica (JB3D); 10. H. zealandica (JBX1); 11.Heterorhabditis sp. (HT390); 12. H. megidis (Microbio); 13. H.bacteriophera (NJ)).

FIG. 2 shows a RAPD gel conducted on DNA from H. zealandica and othercomparative strains. The primer used was OP-F03: 5′-CCTGATCACC-3′ (SEQID NO:2) (Operon Technologies Inc.). (Key: 1. 100 bp DiNA Ladder; 2. H.zealandica (Great Keppel A); 3. H. zealandica (Great Keppel B); 4. H.zealandica (Great Keppel C); 5. H. zealandica (Windsor); 6. H.zealandica (NZH3); 7. H. zealandica (WA Het); 8. Heterorhabditis sp.(JB6); 9. H. zealandica (JB3D); 10. H. zealandica (JBX1); 11.Heterorhabditis sp. (HT390); 12. H. megidis (Microbio); 13.Heterorhabditis sp. (M145); 14. H. bacteriophera (NJ)).

FIG. 3 shows a RAPD gel conducted on DNA from H. zealandica and othercomparative strains. The primer used was OP-X11: 5′-GGAGCCTCAG-3′(SEQ IDNO3) (Operon Technologies Inc.). (Key: 1. 100 bp DNA Ladder; 2. H.zealandica (Great Keppel A); 3. H. zealandica (Great Keppel B); 4. H.zealandica (Great Keppel C); 5. H. zealandica (Windsor); 6. H.zealandica (NZH3); 7. H. zealandica (WA Het); 8. Heterorhabditis sp.(JB6); 9. H. zealandica (JB3D); 10. H. zealandica (JBX1); 11.Heterorhabditis sp. (HT390)).

FIG. 4 provides graphical results of pot assays conducted to assess themortality of Dermolepida achieved by H. zealaiidica strains JB1/X1,JB1/Q and NZH3 and Steinernema glaseri strain NC34.

FIG. 5 provides graphical results of pot assays conducted to assess themortality of C. siguaticoilis achieved by H. zealandica strains JB1/X1and NZH3, S. glaseli strain NC34 and H. bacteriophora HP88.

FIG. 6 provides graphical results of pot assays conducted to assess themortality of A. parvulus achieved by H. zealandica strains JB1/X1 (Per100, 330 and 100 nematodes) and NZH3, and S. glaseri strain NC34.

FIG. 7 provides graphical results of pot assays conducted to assess themortality of L. negatoria achieved by H. zealandica strain JB1/X1 and S.glaseri strain NC34.

FIG. 8 provides graphical results to assess the mortality of adult H.arator achieved with H. zealandica strains JB1/X1, NZH3 and Botany, S.glaseri strain NC34, S. feltiae and S. carpocapsae BW.

FIG. 9 provides graphical results of studies conducted to deterimneLD50s of H. zealandica strains in pot assays against final instar C.signaticollis.

FIG. 10 provides graphical results in field trials of Heterorhabditiszealandica X1 against African black beetle scarab.

EXAMPLE 1 Isolation and Characterisation of H. zealandica Nematodes

Soil samples infested with nematodes were collected from a number offield sites throughout Australia. Using the method of Bedding andAkhurst (1975), nematodes were isolated and subsequently assigned to aspecies on the basis of morphological characterisation (see Wouts(1979)) and DNA analysis (see Tables 1 and 2). Eight of the isolatednematodes were assigned to the species H. zealazidica.

Samples of three of the H. zealandica strains, namely JB1/X1, GKB andJB3D were deposited under the Budapest Treaty with the AustralianGovernment Analytical Laboratories (AGAL), P.O. Box 385, Pymble, NewSouth Wales 2073, Australia. These deposits have been accorded theAccession Nos. 10726, 10727 and 10728, respectively.

Random Amplificatioii of Polymorphic DNA (RAPD) studies were conductedon DNA from the H. zealandica nematodes in accordance with the method ofHashmi, Glazer and Gangler (1996). The results which are presented inFIGS. 1-3, indicate that various of the strains may be seperated on thebasis of their RAPD patterns (e.g. strains JB1/X1 and JB3D can bedistinguished on the basis of their RAPD pattern obtained with theprilner OP-X11).

TABLE 1 Percentage Similarity of Domain 3 DNA Sequences ofHeterorhabditis Strains. (299 bp Region) NZH3 Windsor JBX1 JBQ1 WA HetIrish M145 HT390 Microbio NJ C1 NZH3 100 Windsor 100 100 JBX1 100 100100 JBQ1 100 100 100 100 WA Het 100 100 100 100 100 Irish M145 98.6798.67 98.67 98.67 98.67 100 HT390 98.67 98.67 98.67 98.67 98.67 100 100Microbio 98.33 98.33 98.33 98.33 98.33 99.67 99.67 100 NJ 94.98 94.9894.98 94.98 94.98 96.32 96.32 95.99 100 C1 94.65 94.65 94.65 94.65 94.6595.99 95.99 94.98 99.00 100 Known comparative strains: NZH3 (H.zealandica), Microbio (H. megidis), Irish M145 (“Irish Megidis”), NJ &C1 (H. bacteriophora). Deletions have been included as base pairchanges.

TABLE 2 Percentage Similarity of Internal Transcribed Spacer 1 DNASequences of Heterorhabditis Strains. (805 bp Region) NZH3 Windsor JBX1JBQ1 WA Het Irish M145 HT390 H. hepialus H. marelatus Microbio NZH3 100Windsor 100 100 JBX1 99.88 99.88 100 JBQ1 99.88 99.88 100 100 WA Het99.88 99.88 100 100 100 Irish M145 89.07 89.07 89.19 89.19 89.19 100HT390 88.57 88.57 88.70 88.70 88.70 94.78 100 H. hepialus 81.80 81.8081.80 81.80 81.80 91.24 99.31 100 H. marelatus 81.57 81.57 81.57 81.5781.57 91.01 99.08 99.77 100 Microbio 84.97 84.97 85.09 85.09 85.09 93.6689.69 87.56 87.33 100 Known comparative strains: NZH3 (H. zealandica),Microbio (H. megidis), Irish M145 (“Irish Megidis”). H. marelatus and H.hepialus sequences were extracted from Genbank and their comparisons arebased on a 434 bp overlap of the 805 bp ITS1 region. Deletions have beenincluded as base pair changes.

EXAMPLE 2 Control of Scarabs with H. zealandica Nematodes

A variety of trials were conducted with several strains of H. zealandicaand other nematodes against scarabs. Laboratory based trials included acomparison of strains by dosing a homogeneous selection of larvae withan equivalent number of nematodes of each strain and assessing thenumber of larvae killed (see Comparison Trials), and the determinationof LD50s from pot assays against final instar Cycoceplhala signaticollis(see LD50 Trials).

Comparison Trials

1. Dermolepida

Pot assays using the method of Bedding, Molyneux and Akhurst (1983) (seeLD50 Trials), were conducted using final instar larvae of Dermolepidadosed with H. zealandica strains JB1/X1 and JB1/Q. JB1/X1 achieved 70%kill while JB1/Q had little effect (at 1000 IJs/pot). Subsequentexperiments with the above strains as well as H. zealandica NZH3 and S.glaseri NC34 at the same dose (see FIG. 4) showed that JB1/X1 achievedsuperior kill (over 90% kill after 3 weeks with JB1/X1 versus 57% after3 weeks with S. glaseri NC34, being the best of the others). Controlmortality was too high in tests done on first instar larvae to give avalid result.

2. Cyclocephala

Pot assays were conducted on final instar C. signaticollis larvae withthe NC34 strain of S. glaseri, H. bacteriophora HP88 and the NZH3 andJB1/X1 strains of H. zealandica (at 500 Ijs/pot). The results obtained(see FIG. 5) showed that JB1/X1 achieved superior kill. Subsequentassays comparing JB1/X1 with a range of other strains of H. zealandica(namely, JB3/D, Botany, GKA, GKB, GKC and JB3/F) at a dose of 200IJs/pot showed JB1/X1 to equal (JB3/D, Botany, GKB and GKC) or superior(B3/F and GKA) in killing power. In addition, pot assays comparingJB1/X1 to a range of field collected material at 200 IJs/pot (niamiely,JB4/A to JB4/E, Qld CS9, and JB4/H, all of which are H. zealandica),JB1/X1 was again found to achieve superior kill.

3. Antitrogus parvulus

Pot assays were conducted on final instar Antitrogus parvulus comparingH. zealandica strains JB1/X1 and NZH3 and S. glaseri strain NC34. Theresults (see FIG. 6) showed that NZH3 achieved the best killparticularly in the short term. Further, NZH3 achieved close to itsmaximum level of control in one week whereas JB1/X1 required two weeks.Subsequent assays using a lower dose (500 instead of 1000) resulted inall of H. megidis strain MicroBio H. bacteriophora strain HP88 and H.zealandica strains JB1/X1 and NZH3 achieving a very poor level of kill.

4. Lepidiota negatoria

Pot assays were conducted on final instar Lepidiota negatoria comparingH. zealandica strain JB1/X1 and S. glaseri strain NC34. The results (seeFIG. 7) showed that JB1/X1 achieved over 80% kill with 100 nematodes perlarvae after 2 weeks, whereas NC34 achieved only 35% kill with 1000nematodes after the same period.

5. Heteronychus arator

Pot assays were conducted on adult Heteronychus arator (African BlackBeetle) comparing H. zealandica strains JB1/X1, NC34, NZH3 and Botany,S. feltiae, and S. carpocapsae BW at 1000 IJs/pot. The results (see FIG.8) showed that BW achieved some control while the others wereineffective.

6. Xylotrupes gideon

Preliminary studies have showed that the H. zealacidica strain JB1/X1 ata dose of 1000 IJs/pot, is capable of killing Xylotrupes gideon larvae.

7. Anoplognathus sp.

Preliminary studies have showed that the H. zealandica strain JB1/X1 ata dose of 1000 IJs/pot, is capable of killing Anoploplognathus species.

LD50 Trials with C. signaticollis

Assays to determine the LD50s of the H. zealandica strains Windsor,JB1/X1, GKB, NZH3, GKA, JB6, JB3/D and HT390 (H. sp) against freshlycollected final instar C. signaticollis were conducted in accordancewith the following method:

C. signaticollis larvae (collected from a playing field at theAustralian National University, Canberra, Australian Capital Territory)were exposed individually to nematodes within plastic screw-cap specimenjars (diameter 4.2 cm, height 6 cm) filled to within 1 cm of the topwith approximately 80 g of fine sand, moisture content to about 7%(pF=1.3). The larvae were placed at the bottom of the jars. Nematodeswere introduced in 1 ml of water into a centrally placed well at the top(0.5 cm diameter, 2 cm deep), which was then filled with sand. Numbersof nematodes were estimated by dilution counts. There were 20applications of each dosage for each nematode strain. After 14 daysincubation at a temperature of 23 degrees C. larvae were removed and ifdead were dissected and microscopically examined for neatode infectionin the insect Ringers solution. The LD50 values were computed using theprobit analysis of Finney 1971.

The results are shown in FIG. 9. In particular, FIG. 9 shows the levelof kill achieved by each strain at a range of doses (corrected forcontrol mortality).

These results allowed the calculation of LD50s against C. signaticollisas follows in Table 3:

TABLE 3 LD50s of various strains of Heterorhabditis zealandica againstfinal instar Cyclocephala signaticollis Strain LD50 95% Limits StrainLD90 95% Limits JB3/D 110  57 222 JB3/D 739.7 315.7 14430 JB1/X1 121  65203 JB1/X1 848.7 430.8  3708 WINDSOR 148  83 250 WINDSOR 10004 506.4 4384 GKB 230 109 444 GKB 3755 1473 28650 NZH3 596 340 1052  NZH3 43392062 22781 GKA 562 286 1094  GKA 6041 2427 63009 JB6 3170  1194  47424 JB6 122652 15096 199788676   HT390 4831  1064  2E+08 HT390 65842 90241.29E+20 LD50s and LD90s and confidence limits calculated using theprobit analysis and Fieller procedures in Genstat 5

LD50 Trials with Other Scarab Species

LD50s of H. zealandica strain JB1/X1 against other species of scarabs(i.e. the Japanese beetle, P. japonica) may be determined by thefollowing method.

1. Scarab larvae are exposed individually to nematodes within plasticscrew cap specimen jars (diameter 4.2 cm, height 6.0 cm) filled towithin one cm of the top with 80 g of clean, fine sand carefully mixedwith water to achieve an eveii moisture content of 7% (Pf=1.3). A larvalscarab is placed at the bottom of each specimen jar, sand lightly packedover it and the top screwed on tigltly.

2. Nematodes are introduced in 1 ml of water into a centrally placedwell (0.5 cm diameter, 2 cm deep), which is then filled with sand.

3. Five dosages of IJs are each applied to 20 scarab larvae with afurther 40 larvae as controls (total of 140 larvae). The dosages usedare 10, 33, 100, 330, and 1000 IJ/ml.

4. Larvae are examined after one week and two weeks at 23 degrees C.with live and dead recorded on both occasions.

5. LD50s are calculated using the probit analysis and Fieller procedureswithin the software package, Genstat 5 (Genstat 5, Release 4.1, ThirdEdition, Lawes Agicultural Trust, (IACR-Rothamsted), 1997).

Tables 4 and 5 provides the results of LD50s of H. zealandica againstvarious scarab species using the above method.

TABLE 4 LD50s and LD90s of various scarabs after 2 weeks exposure to H.zealandica JB1/X1 Scarab Species 95% Limits LD50 Adoryphorus couloni 12874 228 Lepidiota negatoria 177 85 317 Antitrogus parvulus 186 75 449Cyclocephala signaticollis 121 65 203 Popillia japonica 115.6* 75 181LD90 Adoryphorus couloni 1396 627 6486 Lepidiota negatoria 964 438 6810Antitrogus parvulus 3402 1009 227541 Cyclocephala signaticollis 848.7430.8 3708 Popillia japonica 1174* 871 5742 *from pooled data

LD50 and LD90 and confidence limits were calculated using the probitanalysis and Fieller procedures in Genstat 5.

TABLE 5 LD50 results from tests on Popillia japonica Lower 95% Upper 95%Rep 1 LD50 After 1 week 5236 681.1 3.94E + 18 After 2 week 182.3 95.42410.9 Rep 2 LD50 After 1 week 147.9 86.81 265.9 After 2 week 76.38 41.01134.9 Pooled reps LD50 After 1 week 412.7 233.2 993.7 After 2 week 115.674.81 180.8 LD90 After 2 weeks 1174 871 5742

EXAMPLE 3 Effectiveness of H. zlalandica Nematodes Against VariousScarabs in the Field

Small scale field trials were conducted by treating small turfed areas,followed by periodic “digging uip” to count live and dead scarabs andlarvae.

1. One trial was made during February 1999 at the Peninsula Golf Club inVictoria (Australia) where there was a heavy infestation of black beetleH. arator, larvae. Four turfed area of 10 m² were treated with H.zealandica JB1/X1 at an amount of 250,000 IJ/m² as part of a randomblock design including various other treatments. Observations revealedthat 50% of larvae in the treated area were killed after two weeks and80% after three weeks (see FIG. 10), whereas there was negligibleneinatode death in the control plots. Separate from this trial, the golfclub superintendent treated larger areas of turf (about 2 hectares) withonly 100,000 IJ/m² and found no bird feeding damage in the treated areabut significant damage nearby.

2. In a second trial, 500,000 IJ/m² were sprayed over 100 m² of aCanberra soccer field (Australian Capital Territory) heavily infestedwith Argentine scarab, C. signaticollis. After eight days, 33% of thenemiatodes were dead, after 23 days 52%, and after thirty days 61% weredead over six sample areas but only 3% were dead in ain area of drysoil. After 68 days no scarabs were found alive and 16 dead in thirteen200 cm² samples of treated area whereas 18 live larvae were found inuntreated areas.

3. Small areas in two Canberra back garden lawns (Australian CapitalTerritory) with very lush grass and severe infestations of C.signaticollis were treated with H. zealandica at one million IJ/m². Inthe first garden, after 11 days, there were 30% nematodes dead in oneplot and after 35 days, 74% nematode death in one plot with 90% death inanother (based on only 19 and 11 larvae respectively). In the secondgarden, no dead scarabs could be found after 20 days, but after 40 daysa total of 42 dead and 4 live scarabs (91%) were found in samples ofthree plots.

4. In late February 1999, 4 areas of turf within a quadrangle at theAustralian National University (Australian Capital Territory) weretreated with H. zealandica at 500,000 IJ/m². After two weeks there was alittle nematode death, after three weeks all average of 48% nematodedeath over three plots, after 4 weeks 81%, and after six weeks 90%nematode death.

The results above indicate that certain strains of H. zealandica may beused as biological control agents for scarabs. Since H. zealandicanematodes may be readily and cost-effectively reared using solid cultureas described by Bedding (1981, 1984); their use in liquid or solidcompositions would appear to offer a viable alternative to lawn scarabcontrol by chemical pesticide spraying.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

References

1. BEDDING, R. A. (1981). Low cost in vitro mass production ofNeoaplectana and Heterorhabditis species (Nematoda) for field control ofinsect pests. Nematologica 27: 109-14.

2. BEDDING, R. A. (1984). Large scale production, storage and transportof the insect parasitic nematodes Neoaplectana spp. and Heterorhabditisspp. Ann. appl. Biol. 104: 117-120.

3. BEDDING, R. A. AND AKHURST, R. J. (1975). A simple technique for thedetection of insect parasitic rhabditid nematodes in soil. Nematologica21: 109-10.

4. BEDDING, R. A., MOLYNEUX, A. S. AND AKHURST, R. J. (1983).Heterorhabditis spp., Neoaplectana spp. and Steinernema Kraussei:Interspecific and intraspecific differences in infectivity for insects.Experimental Parasitology 55: 249-57.

5. BEDDING R. A., M. S. STANFIELD, AND G. W. CROMPTON (1991). Apparatusand Method for Rearing Nematodes, Fungi, Tissue Cultures and The Like,and For Harvesting Nematodes. International Patent Application NoPCT/AU91/00136.

6. HASHMI, G., GLAZER, I. & GAUGLER, R. (1996). Molecular comparisons ofentomnopathogenic nematodes using Random amplified Polymorphic DNA(RAPD) markers. Fundam. appl. Nematol., 18:55-61.

3 1 10 DNA Heterorhabditis zealandica 1 aatcgggctg 10 2 10 DNAHeterorhabditis zealandica 2 cctgatcacc 10 3 10 DNA Heterorhabditiszealandica 3 ggagcctcag 10

What is claimed is:
 1. A composition comprising an amount of anentomopathogenic nematode selected from the group of H. zealandicastrains consisting of strains JB1/X1, GKB and JB3D, optionally inadmixture with a suitable agricultural and/or horticultural carrier. 2.The composition of claim 1, wherein the amount of the entomopathogenicnematode is an amount in the range of about 50 to 10,000 nematodes/ml ofcomposition.
 3. The composition of claim 2, wherein the amount of theentomopathogenic nematode is an amount in the range of about 500 to10,000 nematodes/ml of composition.
 4. A method for controlling apopulation of larval and/or pupal scarabs in an affected area, saidmethod comprising applying to said area a composition in accordance withclaim 1, thereby the population of the larval and/or pupal scarabs iscontrolled.
 5. The method of claim 4, wherein the composition is appliedto the affected area so as to provide a dose of 50,000 to 1,000,000IJ/m².
 6. The method of claim 5, wherein the composition is applied tothe affected area so as to provide a dose of 100,000 to 500,000 IJ/m².7. The method of claim 4, wherein the composition is applied to theaffected area at dusk.
 8. The method of claim 4, wherein the method isfor controlling a population of larval and/or pupal scarabs selectedfrom the group of scarab species consisting of Cyclocephalasignaticollis, Heteronychus arator, Adoryphorus couloni, Antitrogusmorbillosus, Anoplognathus porosus, Ataenius imparalis, Sericesthisgeminata, S. pruinosis, S. nigrolineata, Scityla sericans, Saulostomusvillosus, Aphodius tasmaniae, Heteronyx spp, Rhopoea magnicornis,Popillia japonica, Cyclocephala borealis, C. hirta, C. parallela,Melolontha melolontha, Anomala aenea, Phyllophaga phyllophaga, P.hirticula, Phyllopertha horticola, Haplididia etrusca, Maladea matrida,Costelytra zealandica, Amphimallon solstatialis and Ligyrus subtropicus.9. The method of claim 8, wherein the method is for controlling apopulation of larval and/or pupal scarabs of the species of C.signaticollis.
 10. The method of claim 8, wherein the method is forcontrolling a population of larval and/or pupal scarabs of the speciesof P. japonica.
 11. An isolated nematode selected from the H. zealandicastrains designated JB1/X1, GKB and JB3D.
 12. A process for producing acomposition for controlling larval and/or pupal scarabs comprising: (i)subjecting one or more candidate entomopathogenic nematodes of thespecies Heterorhabditis zealandica to a 14-day pot assay against finalinstar Cyclocephala signaticallis larvae and/or final instar Popillajaponica larvae; (ii) selecting an entomopathogenic nematode which hasan LD₅₀ value of less than 300 IJ as measured by the 14-day pot assay;and (iii) admixing the entomopathogenic nematode of step (ii) with asuitable agricultural or horticultural carrier.
 13. The process of claim12 wherein the entomopathogenic nematode is a strain of H. zealandicawhich has an LD₅₀ value of less than 175 IJ as measured by the 14-daypot assay against final instar C. signiticollis larvae and/or finalinstar P. japonica larvae.
 14. The process of claim 12 wherein theentomopathogenic nematode is selected from the group of H. zealandicastrains consisting of strains JB1/X1, GKB and JB3D.
 15. The process ofclaim 12 wherein the amount of the entomopathogenic nematode admixedwith the carrier is in the range of about 50 to 10,000 nematodes/ml. 16.A method for controlling a population of larval and/or pupal scarabs inan affected area, said method comprising applying to said area acomposition produced in accordance with claim 12, thereby the populationof the larval and/or pupal scarabs is controlled.
 17. The method ofclaim 16 wherein the composition is applied to the affected area so asto provide a dose of 50,000 to 1,000,000 IJ/m².
 18. The method of claim16 wherein the composition is applied to the affected area at dusk. 19.The method of claim 16 wherein the method is for controlling apopulation of larval and/or pupal scarabs selected from the group ofscarab species consisting of Cyclocephala signaticollis, Heteronychusarator, Adoryphorus couloni, Antitrogus morbilliosis, Anagplognathusporosus, Ataenius imparalis, Sericesthis geminant, S. pruinosis, S.noigrolineata, Scityla sericans, Saulostomus villosus, Aphodiustasmaniae, Heteronyx spp, Rhopoea magnicornis, Popillia japonica,Cyclocephala borealis, C. hirta, C. parallela, Melolontha melolontha,Anomala aenea, Phyllophaga phyllophaga, P. hirticula, Phyllopherthahorticola, Haplididia etrusca, Maledea matrida, Costelytra zealandica,Amphimallon solstatialis, and Lygyrus subtropocus.